Liangxue Jiedu Huayu Formula improves liver function of mice with acute-on-chronic liver failure by inhibiting excessive activation of the cGAS-STING signaling pathway

doi:10.12122/j.issn.1673-4254.2024.12.04

Abstract

Abstract:

Objective To explore the role of the cGAS-STING signaling pathway in the therapeutic mechanism of Liangxue Jiedu Huayu Formula (LXJDHYF) for acute-on-chronic liver failure (ACLF) in mice. Methods Thirty C57BL/6 mice were randomly divided into blank control group, model group, low- and high-dose LXJDHYF groups, and H151 (a specific cGAS-STING pathway inhibitor) group (n=6). In all but the control group, the mice were treated with CCl₄ to induce liver cirrhosis followed by intraperitoneal injections of lipopolysaccharide and D-amino galactose to establish mouse models of ACLF. After the treatments, the mouse livers were collected for HE and TUNEL staining, and serum levels of ALT, AST and TBil were determined. In bone marrow-derived macrophages (BMDMs) and liver tissues of ACLF mice, the expressions of cGAS-STING signaling pathway-related mRNAs including IFN‑β, ISG15, IL-6 and TNF-α were determined with RT-qPCR, and the phosphorylation levels of IRF3 and STING proteins were investigated using Western blotting. Results Compared with the mice in the model group, the LXJDHYF-treated mice exhibited milder hepatocyte necrosis and inflammatory cell infiltration in the liver with significantly reduced hepatocyte apoptosis. LXJDHYF treatment also significantly lowered serum levels of ALT, AST, TBil, IL-6 and TNF-α in ACLF mice and effectively suppressed the expressions of cGAS-STING signaling pathway-related mRNA in both the BMDMs and the liver tissues and the phosphorylation of IRF3 and STING proteins in the BMDMs. Conclusion LXJDHYF can significantly improve liver function and attenuate inflammation in ACLF mice possibly by inhibiting excessive activation of the cGAS-STING signaling pathway.

Key words: acute-on-chronic liver failure, cGAS-STING signaling pathway, interferon?β, interferon stimulating gene 15, Liangxue Jiedu Huayu Formula

Qiao TANG, Chao ZHOU, Zhaofang BAI, Qing YAO, Simin CHEN, Xinru WEN, Zhaoyun HE, Jin ZHANG, Ruisheng LI, Man GONG. Liangxue Jiedu Huayu Formula improves liver function of mice with acute-on-chronic liver failure by inhibiting excessive activation of the cGAS-STING signaling pathway[J]. Journal of Southern Medical University, 2024, 44(12): 2291-2299.

Figures/Tables 8

Tab.1 Primer sequences for RT-qPCR

Gene	Sequence
β-ACTIN	P1	GGCTGTATTCCCCTCCATCG
β-ACTIN	P2	CCAGTTGGTAACAATGCCATGT
IFN-β	P1	TCCGAGCAGA GATCTTCAGGAA
IFN-β	P2	TGCAACCACCACTCATTCTGAG
TNF-α	P1	GGGCAGTTAGGCATGGGAT
TNF-α	P2	TGAGCCTTT TAGGCTTCCCAG
IL-6	P1	CACTTCA CAAGTCGGAGGCT
IL-6	P2	CTGCAAGTGCATCATCGTTGT
ISG15	P1	GGTGTCCGTGACTAACTCCAT
ISG15	P2	CTGTACCACTAG CATCACTGTG

Fig.1 HE staining (Original magnification: ×200) and TUNEL staining (×20) for examining pathologies and hepatocyte apoptosis in mouse livers (A) and comparison of TUNEL-positive cell percentages among the groups (B). ***P<0.001 vs model group.

Fig.2 Serum levels of ALT (A), AST (B) and TBil (C) of the mice in each group. ***P<0.001 vs model group.

Fig.3 ATP assay for evaluating cytotoxicity of different concentrations of LXJDHYF in BMDMs. ***P<0.001 vs 0 mg/mL.

Fig.4 Effects of different concentrations of LXJDHYF on expressions of IFN-β (A), ISG 15 (B), TNF-α (C) and IL-6 mRNAs (D) in BMDMs with ISD-induced cGAS-STING signaling pathway activation. E-H: Expressions of IFN-β (E), ISG 15 (F), TNF-α (G) and IL-6 mRNAs (H) in BMDMs pre-treated with LXJDHYF (1 mg/mL) prior to stimulation with different STING agonists. *P<0.05, **P<0.01, ***P<0.001 vs 0 mg/mL.

Fig.5 Western blotting for detecting phosphorylation levels of STING and IRF3 proteins in BMDMs with ISD-induced cGAS-STING signaling pathway activation and treated with different concentrations of LXJDHYF (A-C). D-F: Phosphorylation levels of STING and IRF3 proteins in BMDMs pre-treated with LXJDHYF (1 mg/mL) and then stimulated with different STING agonists. *P<0.05, **P<0.01, ***P<0.001 vs 0 mg/mL.

Fig.6 Expressions of IFN-β (A), ISG15 (B), TNF-α (C) and IL-6 mRNAs (D) in the liver tissues of the mice in each group. ***P <0.001 vs model group.

Fig.7 Serum levels of IL-6 (A) and TNF-α (B) of the mice in each group. *P<0.05, **P<0.01, ***P<0.001 vs model group.

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